The present invention relates to a method and an apparatus for ROI-scan with a high temporal resolution, preferably the method is used in confocal scanning microscopy.
In confocal scanning microscopy, a specimen is scanned with a focused light beam; this is generally achieved by tilting two mirrors arranged in the beam path of the confocal scanning microscope. The focus of the light beam is thereby moved in the focal plane, the deflection directions of the light beam most often being arranged perpendicular to one another so that, for example, one mirror deflects the beam in the X direction and another mirror deflects the beam in the Y direction. The motion or tilting of the mirrors is usually brought about with the aid of galvanometer actuating elements. Special control devices connected to the galvanometer actuating elements provide a position signal of the light beam on the specimen.
The German patent Application DE 198 29 981 discloses a method which involves coupling laser light of different spectral ranges in an, in at least two coordinates diverted microscope beam path, and directing the laser light successively on places of a test. The test is supplied in at least one plane, place for place and line for line with the laser light, and an image of the sampled plane is generated from reflected and/or emitted light. The spectral composition and/or the intensity of the laser light is changed while the scanning is continued, and at least two adjacent points of the test are supplied with light of different spectral characteristics and/or different intensity. An Independent claim is provided for a laser-scanning microscope implementing the method. The above mentioned method applies the same intensity in the scan direction and in the opposite scan direction. In other words the region of interest is illuminated with the same wavelength and/or illumination condition in the scan direction and in the opposite scan direction. A detection of the influence of the illumination condition on the region of interest is detectable only in the successive frame. For some biological processes the delay between illumination and detection is to long.
It is therefore the object of the present invention to describe a method which improves the time resolution drastically and allows an easy and specimen related change of the illumination conditions.
The above object is achieved by a method for scanning a specimen with an illumination light beam of at least one light source comprising the steps of:
defining at least one region of interest (ROI) in a scan frame of the specimen;
providing a first plurality of first scan lines and a second plurality of second scan lines by the intersection of the scan pattern with the at least region of interest;
illuminating the specimen in the at least one region of in the first scan line with first illumination conditions wherein the region of the sample outside the region of interest is illuminated with second illumination conditions; and
illuminating the specimen in the at least one region of interest in the second scan line with third illumination conditions wherein the region of the sample outside the region of interest is illuminated with fourth illumination conditions.
It is a further object of the present invention to provide an apparatus for ROI-Scan with high local resolution which improves the time resolution drastically and allows an easy and specimen related change of the illumination conditions.
The above object is accomplished by an apparatus for ROI-scan with high temporal resolution of a specimen, comprising:
at least one light source for generating an illumination light beam to be scanned by a scanning device across the specimen in a scan pattern wherein the intersection of the scan pattern and at least one region of interest defining a first plurality of first scan lines and a second plurality of second scan lines;
means for adjusting illumination conditions of the at least one light source wherein the means are positioned in the illumination beam path prior to the scan device, and
control means connected to the scan device and the means for adjusting the illumination conditions in response to the position of the light beam on the specimen.
The advantage of the first embodiment of the invention is that the plurality of first scan lines is substantially superimposed to the plurality of second scan lines and the scan direction of the first scan lines and the second scan lines is unidirectional. In an other embodiment the plurality of first scan lines is substantially parallel to the plurality of second scan lines and the scan direction of the first scan lines and the second scan lines is opposite to each other.
The first illumination conditions and the third illumination conditions are the same wavelength and differ from the second illumination conditions and fourth illumination conditions both of which are the same wavelength. The illumination condition according to the above context is defined by the wavelength xcex. The first and third illumination conditions are defined by a first wavelength xcex1, and the second and fourth illumination condition are defined by a second wavelength xcex2. An AOTF (acoustooptical tunable filter), EOM (electrical optical modulator) or AOM (acoustical optical modulator) is used to switch between the two wavelengths. The switching is dependent from the position of the scanning light beam within the scanned frame of a specimen.
The means for adjusting illumination conditions is an AOTF or AOM which switches the illumination condition between a plurality of intensity levels for one specific wavelength. In a further embodiment the first illumination condition is defined by a first illumination intensity level, the second illumination condition is defined by a second illumination intensity level, the third illumination condition is defined by a third illumination intensity level and the fourth illumination condition is defined by a fourth illumination intensity level.
An additional requirement for the illumination level is, that the first illumination intensity level is greater than the second illumination intensity level and the signals of the first and second illumination intensity level are displayed in a first representation on a display and the third illumination intensity level is equal to the fourth illumination intensity level and the signals of the third and fourth illumination intensity level are displayed in a second representation on the display.
An other inventive distribution of the intensity levels on the scanned specimen is that the first illumination intensity level in a first region of interest is different and greater than the second illumination intensity level and the signals of the first and second illumination intensity level are displayed in a first representation on a display and the third illumination intensity level is smaller than the fourth illumination intensity level and the signals of the third and fourth illumination intensity level are displayed in a second representation on the display.
It is especially advantageous to have the possibility to switch between at least two different laser intensities. In the scan direction 5% of the laser intensity is applied outside the region of interest and 95% of the laser intensity are applied within the region of interest. In the opposite scan direction 5% of the laser intensity are applied outside the region of interest and 0% of the laser intensity are applied within the region of interest. This allows a fast determination of an optimum parameter set for the illumination which eliminates a too high degree of bleaching. The optimum parameter set is the illumination condition which will be applied to the at least one region of interest and/or to the area of the specimen outside the region of interest. It is a further advantage that the with the inventive method and apparatus different parameter sets can be applied to different regions of the specimen.
In a combination the background of a frame may be illuminated with wavelength and illumination intensity level which are different from the wavelength and illumination intensity level within the region of interest. For example the background is illuminated with an illumination intensity level of 5% at a wavelength of 488 nm. The region of interest is illuminated with an illumination intensity level of 100% at a UV wavelength. Moreover, a plurality of regions of interest may be defined all of which may be subjected to different illumination conditions.
The inventive method and apparatus are especially suitable for fast occurring biological processes like diffusion of molecules, communication between living cells, determination of physiological parameters and the determination of membrane potentials, pH-levels, calcium levels or releasing of caged compounds.
Further advantageous embodiments of the invention are apparent from the dependent claims.